CN107987144B

CN107987144B - Centipede polypeptide SLP _ SstX as well as encoding gene and application thereof

Info

Publication number: CN107987144B
Application number: CN201711339873.5A
Authority: CN
Inventors: 赖仞; 杨仕隆; 罗雷
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2020-05-15
Anticipated expiration: 2037-12-14
Also published as: CN107987144A

Abstract

The invention relates to a centipede polypeptide SLP _ SstX as well as a coding gene and application thereof, belonging to the technical field of biomedicine. The invention provides a centipede polypeptide SLP _ SstX, the whole amino acid sequence of the centipede polypeptide SLP _ SstX is shown as SEQ ID NO. 1. The centipede polypeptide SLP _ SstX provided by the invention has the functions of inhibiting KCNQ family channels and mediating a cardiovascular system, a nervous system and a respiratory system, and can be used as a probe template for researching the KCNQ channels and explaining clinical symptoms of centipede bites.

Description

Centipede polypeptide SLP _ SstX as well as encoding gene and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a centipede polypeptide SLP _ SstX as well as a coding gene and application thereof.

Background

The centipede sanderi is a traditional Chinese medicine in China and has the effects of toxin relieving, cancer resisting, wind calming and spasmolysis.

Centipede (s. subspinipes mutilans l. koch) is an approximate species of centipede (scolopendra subspinipes) and is mainly distributed in china and japan, and is common in the middle and lower reaches of the Yangtze river in China. The first pair of feet of centipedes has evolved for a long time to form a chela which is in a hook shape and sharp, the hook end of which is provided with a poisonous gland mouth, and the outside of which is coated by hard chitin. The centipede has poisonous glands for secreting and storing the venom in the poisonous chela, the poisonous glands are distributed in a vesicle shape, and surrounding vesicles can be understood as sub-poisonous glands, so that larger space for storing the venom is searched for by poisonous gland cells outside cells. The centipede toxin is colorless and transparent viscous liquid secreted by poison glands, and can be paralyzed and even killed when injected into a hunting object. They have evolved the ability to adapt to prey, such as fish, insects, reptiles, mollusks, amphibians, and even mammals, all of which are prey subjects (Undheim, e.a., and King, g.f. (2011) On the innovative system of centralized animals (Chilopoda), a connected group of innovative animals, toxicon 57, 512-. The centipede has the characteristics of pain, burning, itching, erythema, congestion, inflammation, blistering, subcutaneous hemorrhage, edema, epidermal necrosis and desquamation after being injured by the centipede, and severe cases of headache, nausea, vomiting, fever, irregular heart rate and breathing, lymphadenitis, tissue necrosis, vasospasm, coma and the like, and even death. Centipede toxins have been reported to cause rhabdomyolysis, myocardial ischemia, and even myocardial infarction (Yildiz, a., bicegru, s., Yakut, n., Bilir, c., Akdemir, r., andakili, a. (2006) ingredient myolysis in a healthy human being spent by our nutritional medium j: EMJ 23, e 30).

Although people encounter centipedes and suffer from the injuries, there are few studies on the composition and action mechanism of centipede venom, what substances in the venom can produce the physiological reactions explained above? Therefore, the exploration of functional active molecules of the centipede venom is helpful for understanding the pathogenesis of clinical symptoms and the pathogenesis, and can lead people to intervene in the centipede bite in a targeted manner, thereby avoiding the injury of the centipede bite. Most of domestic and foreign researches on centipede poison stay at the level of crude poison research, and researches on effective components and pharmacological activity of the centipede poison are still deep. The centipede venom contains extremely complex chemical components which can be mainly divided into proteins and non-proteins, and the proteins can be further divided into enzymes and non-enzymes. In addition, the centipede feed also contains a large amount of polypeptide substances which play an important role in the predation and defense processes of the centipedes.

The KCNQ potassium channel is a voltage-gated (Kv) ion channel, which comprises 5 family members: KCNQ 1-5. KCNQ1 is mainly expressed in peripheral tissues and heart, while KCNQ2-5 is widely expressed in nervous system, muscular system and cardiovascular system. Current research has found that mutations and disease associated with four KCNQ channels in five subfamily members of KCNQ. The mutation of KCNQ1 is related to long QT syndrome, some is accompanied by deafness, and the most obvious expression of the long QT syndrome is prolongation of QT interval of electrocardiogram, so that the mutation is also accompanied by arrhythmia, sudden cardiac death and the like. Mutations in the KCNQ2 channel and KCNQ3 result in benign familial infantile convulsions, some of which are also accompanied by hyperexcitability of peripheral nerves, while benign familial infantile convulsions are generally 2-4 days after birth of infants, so also known as "third day convulsions", 11% of patients later develop neurological disorders such as epilepsy (Bal, M., Zhang, J., Hernandez, C.C., Zaika, O., and Shapiro, M.S. (2010) Ca2+/calmodulin disorders AKAP79/150 interactions with NQ (M-Type) K + channels. the journal of Neuroscience: the clinical J.of Society for Neuroscience30, 2311; Brown, D.2323, A. G.M. and 9. 1195. the Japanese patent publication No. (1185) of cosmetics). Mutations in the KCNQ4 channel cause congenital deafness, a hearing disorder that occurs in infants at birth or after birth. Only the KCNQ5 channel has not been reported to be associated with disease, but it has been reported that KCNQ5 is associated with the formation of resting potential (Brueggemann, L.I., Haick, J.M., Neuburg, S., Tate, S., Randhawa, D., Cribbs, L.L., and Byron, K.L (2014) KCNQ (Kv7) positional channel activity as bridges: communication with both a 2-acquisition admixture of phases of tensile analysis. American journenals of physical analysis. Lung cellular and molecular analysis 306, L476-486).

In view of the important role played by the KCNQ family in the various physiological functions responsible for chloride secretion and cardiac action potential repolarization (KCNQ1), coronary circulation and reactive hyperemia (KCNQ4) and cerebral neuron excitation (KCNQ2, 3, 4) (Jentsch, T.J. (2000) neurological KCNQ potassiums channels: physiology and role indication. Nature reviews. neurological 1, 21-30), while some of the symptoms described in clinical case reports of centipede bites, such as tissue necrosis, vasospasm, Acute hypertension and myocardial ischemia, etc. (Undheim, E.A., and King, G.F. (2011) On the vascular system of centipedes (Chilopada), a saturated of p of animals, Yang. 23, Ak J. Ak. 12. environmental systems, Ak. 23. 12. Ak. J. 12. environmental group, Ak. 21-30), l. (2003) Antimicrobial and cytolytic peptides of venous organisms. cellular and molecular life sciences: CMLS 60, 2651-2668; ozsarac, m., karcomiglu, o., Ayrik, c., Somuncu, f., and Gumrukcu, s. (2004) ace clock bichemia followingcentrode evolution: case report and review of the performance, Wilderness & environmental media 15, 109-112; senthilkularan, s., meenkshisundaram, r., Michaels, a.d., Suresh, P., and third aikolunshubranian, P. (2011) effect ST-segment evaluation muscle interaction from a central bit. journal of cardiovascular disease research 2, 244-246), which is highly similar to clinical symptoms resulting from KCNQs dysfunction, we concluded that the symptoms described in these clinical case reports and may be related to the activity of KCNQs channels. However, the prior art does not report the relationship between the centipede toxin and KCNQs in detail, and does not report the centipede toxin acting on KCNQs family channels. Research into the clinical symptoms and treatment of centipede bites has been hampered.

Disclosure of Invention

The invention aims to provide a centipede polypeptide SLP _ SstX as well as a coding gene and application thereof. The centipede polypeptide SLP _ SstX provided by the invention has the functions of inhibiting KCNQ family channels and mediating a cardiovascular system, a nervous system and a respiratory system, can be used as a probe template for exploring KCNQ channels and explaining clinical symptoms of centipede bites, and further develops and relieves the application of a medicament template for centipede bites.

The invention provides a centipede polypeptide SLP _ SstX, the whole amino acid sequence of the centipede polypeptide SLP _ SstX is shown as SEQ ID NO. 1.

The invention also provides a gene for coding the centipede polypeptide SLP _ SstX in the technical scheme, which comprises a mature peptide part corresponding gene and a signal peptide part corresponding gene, and the nucleotide sequence of the centipede polypeptide SLP _ SstX gene is shown as SEQ ID NO. 2.

Preferably, the nucleotide sequence of the corresponding gene of the centipede polypeptide SLP _ SstX mature peptide part is shown as SEQ ID NO. 3.

The invention also provides an amplification method of the centipede polypeptide SLP _ SstX gene in the technical scheme, which comprises the following steps:

1) extracting total RNA by taking venom glands of centipedes with few thorns as templates;

2) purifying mRNA from the total RNA obtained in the step 1) to obtain mRNA;

3) carrying out reverse transcription on the mRNA obtained in the step 2) to obtain a cDNA library;

4) amplifying a corresponding gene of a centipede polypeptide SLP _ SstX mature peptide part by using a first degenerate primer pair by taking a cDNA library as a template; amplifying a corresponding gene of the centipede polypeptide SLP _ SstX signal peptide part by using a second degenerate primer pair by taking the cDNA library as a template; obtaining the complete sequence of the gene of the centipede polypeptide SLP _ SstX;

the nucleotide sequence of the first degenerate primer pair is shown as SEQ ID NO.4 and SEQ ID NO. 5;

the nucleotide sequence of the second degenerate primer pair is shown as SEQ ID NO.6 and SEQ ID NO. 7;

wherein, in the sequence of the first degenerate primer pair, R ═ A or G, Y ═ C or T, N ═ A or C or G or T.

The invention also provides a KCNQ family channel inhibitor, which comprises the centipede polypeptide SLP _ SstX and auxiliary materials in the technical scheme.

Preferably, the adjuvant comprises a sterile aqueous solution of 0.9% sodium chloride.

The invention also provides application of the centipede polypeptide SLP _ SstX in preparing a medicine for relieving centipede bite.

The invention provides a centipede polypeptide SLP _ SstX. The centipede polypeptide SLP _ SstX provided by the invention is a single-chain polypeptide encoded by centipede oligochaeta gene, and the molecular weight of the polypeptide is 6017.6 daltons. The centipede polypeptide SLP _ SstX is composed of 53 amino acids, and the whole sequence is as follows: EVIKKDTPYKKRKFPYKSECLKACATSFTGGDESRIQEGKPGFFKCTCYFTTG are provided. The centipede polypeptide SLP _ SstX provided by the invention has the functions of inhibiting KCNQ family channels and mediating a cardiovascular system, a nervous system and a respiratory system no matter whether the centipede polypeptide SLP _ SstX is naturally synthesized or artificially synthesized, can be used as a probe template for exploring the KCNQ channels and explaining clinical symptoms of centipede bites, further develops and relieves the application of a centipede bite medicine template, and has the advantages of simple sequence and convenience in synthesis.

Drawings

FIG. 1 shows the cDNA, amino acid sequence and polypeptide tertiary structure of centipede polypeptide SLP _ SstX of the invention 1, wherein A in FIG. 1 is the cDNA sequence and amino acid sequence of SLP _ SstX; b in FIG. 1 is the mass spectrometric detection result and disulfide bond pairing; c in FIG. 1 is a solution state tertiary structure;

FIG. 2 is a graph showing the inhibitory activity of the centipede polypeptide SLP _ SstX on KCNQ family channels, provided in example 4 of the invention, wherein A in FIG. 2 is SLP _ SstX inhibiting human KCNQ4 channel; b in FIG. 2 is a concentration-dependent curve of SLP _ SstX inhibiting KCNQ family channels;

FIG. 3 is a graph showing the effect of centipede polypeptide SLP _ SstX on vasoconstriction of thoracic aorta, as provided in example 4 of the present invention; wherein, A in figure 3 is the effect of crude drug, phenylephrine and acetylcholine on thoracic aorta vasoconstriction; b in FIG. 3 is crude drug, phenylephrine and acetylcholine without SstX; c in FIG. 3 is SstX, phenylephrine, and acetylcholine; d in FIG. 3 is crude toxin and Retigabine (RTG); e in FIG. 3 is SstX and RTG; the concentration curves of RTG versus KCNQ4 for F in FIG. 3 under 5 μ M Sstx conditions;

FIG. 4 is a graph showing the results of the centipede polypeptide SLP _ SstX causing cardiovascular system diseases provided in example 4 of the present invention; wherein, A in figure 4 is the effect of tail vein injection physiological saline and centipede crude drug (30mg/kg) on the systolic pressure (SBP) and diastolic pressure (DBP) of mice; b in FIG. 4 is physiological saline and SstX (0.5 mg/kg); c in FIG. 4 is the effect of intravenous SstX (0.1mg/kg, 0.01mg/kg) on the Systolic (SBP) and Diastolic (DBP) pressure of the macaque; d in FIG. 4 is SstX (0.1mg/kg) and RTG; e in FIG. 4 is a representative electrocardiogram of macaques injected with saline or Sstx;

FIG. 5 is a graph showing the results of the centipede polypeptide SLP _ SstX affecting the nervous system and respiratory system provided in example 4 of the present invention; a in FIG. 5 is the neural firing response of CA1 pyramidal neurons before and after (A) SstX (10. mu.M) treatment; b in FIG. 5 is the firing effect of SstX (10 μ M) and Linopiridine (100 μ M) treatments on neurons; effect of SstX (10. mu.M) and Linopiridine (100. mu.M) treatment on hippocampal acetylcholine release in FIG. 5C is the effect of SstX (2mg/kg) treatment on rat respiration in FIG. 5; e in fig. 5 is the effect of SsTx treatment on rat respiratory rate; f in fig. 5 is the effect of SsTx treatment on rat respiratory amplitude; g in FIG. 5 is the effect of coarse toxicity and RTG on lung bronchoconstriction; h in fig. 5 is the effect of SsTx and RTG on lung bronchoconstriction.

Detailed Description

The invention provides a centipede polypeptide SLP _ SstX, the whole amino acid sequence of the centipede polypeptide SLP _ SstX is shown as SEQ ID NO. 1. In the invention, the centipede polypeptide SLP _ SstX is a single-chain polypeptide encoded by centipede oligochaeta gene, the molecular weight is 6017.6 daltons, and the amino acid complete sequence of the centipede polypeptide SLP _ SstX is specifically as follows:

the invention also provides a gene for coding the centipede polypeptide SLP _ SstX in the technical scheme, which comprises a mature peptide part corresponding gene and a signal peptide part corresponding gene, and the nucleotide sequence of the centipede polypeptide SLP _ SstX gene is shown as SEQ ID NO. 2. In the present invention, the cDNA corresponding to the gene encoding the centipede polypeptide SLP _ SsTx consists of 228 nucleotides, including a mature peptide portion-corresponding gene and a signal peptide portion-corresponding gene, which have the sequence from 5 'end to 3' end:

in the invention, the nucleotide sequence of the corresponding gene of the centipede polypeptide SLP _ SstX mature peptide part is shown as SEQ ID NO.3, and the sequence from 5 'end to 3' end is as follows:

namely, the nucleotide sequence corresponding to the mature peptide is the 70 th to 228 th nucleotides of the SEQ ID NO.2 sequence: SEQ ID NO. 3. Post-translational modification of the SLP _ SsTx mature peptide cleaves off the signal peptide portion (atggagaaaaaaattattttcctggttttccttgttgcgcttttggcacttccgggattcatttcaact). The gene SEQ ID NO.2 for coding the centipede polypeptide SLP _ SstX cuts off signal peptide in the process of biologically synthesizing the centipede polypeptide SLP _ SstX to obtain the centipede polypeptide SLP _ SstX mature peptide.

2) purifying mRNA from the total RNA obtained in the step 1) to obtain mRNA;

4) amplifying a corresponding gene of a centipede polypeptide SLP _ SstX mature peptide part by using a first degenerate primer pair by taking a cDNA library as a template; amplifying a corresponding gene of the centipede polypeptide SLP _ SstX signal peptide part by using a second degenerate primer pair by taking the cDNA library as a template; obtaining the complete sequence of the gene in the technical scheme;

the nucleotide sequences of the first degenerate primer pair are shown as SEQ ID No.4(5 'GARGTNATHAARAARGAYACN 3') and SEQ ID No.5(5 'AAGCAGTGGTATCAACGCAGAGTACT 3');

the nucleotide sequences of the second degenerate primer pair are shown as SEQ ID NO.6(5 'CGTTTTTGAAAAGTTGTAGTA 3') and SEQ ID NO.7(5 'TGTTATTTTACCACTGGTTAA 3');

The present invention is not particularly limited to the methods of total RNA extraction, mRNA purification, mRNA reverse transcription, and cDNA library construction, and may be carried out using a conventional kit known to those skilled in the art. The gene amplification system and conditions of the present invention are not particularly limited, and those well known to those skilled in the art or those described in the general kit instructions may be used.

The synthetic method of the centipede polypeptide SLP _ SstX is not particularly limited, and comprises a biosynthesis method and a chemical synthesis method, the biosynthesis method of the centipede polypeptide SLP _ SstX is not particularly limited, and a conventional protein expression system well known by a person skilled in the art can be adopted, such as an escherichia coli prokaryotic expression system, a yeast or insect cell eukaryotic expression system; chemical synthesis of the centipede polypeptide SLP _ SstX adopts a PerSeptive biosynthesis system, combines an Fmoc/tert-butyl strategy and an HOBt/TBTU/NMM method to synthesize linear SLP _ SstX.

In the present invention, the centipede polypeptide SLP _ SstX chemical synthesis method and the solution condition folding method preferably comprise the following steps: the complete sequence was synthesized using an automated polypeptide synthesizer (433A, applied biosystems) based on the amino acid sequence of the centipede polypeptide SLP _ SstX mature peptide. The desalted and purified by reverse phase high performance liquid chromatography (RP-HPLC) reverse phase column C18 chromatography and was determined to be greater than 95% pure. Linear SLP _ SsTx was folded using oxidized glutathione (GSSG) under solution conditions, and its disulfide bonds were oxidized to form the native tertiary conformation. The molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). SLP _ SstX with native tertiary structure was used for functional detection. The centipede polypeptide SLP _ SstX has a compact tertiary structure and obvious polarity, arginine at the 12 th position and lysine at the 13 th position form an obvious positive charge surface, and the C end of the polypeptide is relatively flexible.

In the present invention, the method for detecting the tertiary structure of the centipede polypeptide SLP _ SstX preferably comprises the following steps:

600MHz high field nuclear magnetic resonance (600-MHz Bruker AV600) was used to study the tertiary structure of the centipede polypeptide SLP _ SstX. Sample preparation conditions were 500. mu.L of 4mM SLP _ SstX, solution conditions 90% PBS/10% D at pH 6.5₂And O. All nuclear magnetic data obtained were analyzed with NMRPipe/NMRDraw software and spark program. Discovery is used to show the final three-dimensional architectural diagram.

In the present invention, the method for separating and purifying the centipede polypeptide SLP _ SstX preferably comprises the following steps:

and (3) primarily separating and purifying the collected centipede venom by using a gel chromatography column Sephadex G-50, collecting 10000Da polypeptide neurotoxin components, and freeze-drying. Separating the polypeptide neurotoxin component by reverse high performance liquid chromatography (RP-HPLC) and C18 column, and purifying to obtain centipede polypeptide SLP _ SstX. In the invention, the gel chromatographic column Sephadex G-50 is preferably separated and purified by using 0.1M phosphate buffer solution (pH 7.4), and the chromatographic speed is preferably 3ml/10 min; in the present invention, the separation conditions of the polypeptide neurotoxin components are preferably gradient elution with elution system composed of water (containing 0.1% trifluoroacetic acid) and acetonitrile (containing 0.1% trifluoroacetic acid), and the elution speed is 0.7 ml/min.

The invention also provides a KCNQ family channel inhibitor, which comprises the centipede polypeptide SLP _ SstX and auxiliary materials in the technical scheme. In the present invention, the adjuvant preferably comprises a 0.9% sterile aqueous solution of sodium chloride. In the present invention, the half inhibitory concentration of SLP _ SsTx in the inhibitor is preferably 2.52 ± 0.17 micromolar.

The centipede polypeptide SLP _ SstX and the coding gene and application thereof are described in further detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

Scolopendra mutilans polypeptide SLP _ SstX gene clone:

I. extracting total RNA of the centipede mutilans venom glands:

A. the venom gland tissue of the scolopendra mutilans is taken out and stored in liquid nitrogen, 500mg of the frozen venom gland tissue is taken out, 10mL of total RNA extraction buffer (Trizol solution, GIBCOBRL company, USA) is added, and the mixture is placed in a 20mL glass homogenizer for homogenization for 30 minutes.

Adding an equal volume of phenol/chloroform solution, shaking and mixing uniformly, standing at room temperature for 10 minutes, centrifuging at 4 ℃ and 12000rpm for 10 minutes, and removing precipitates.

Adding equal volume of isopropanol into the supernatant, standing at-20 deg.C for 10min, centrifuging at 12000rpm at 4 deg.C for 10min, washing the precipitate with 75% ethanol for 3 times, and air drying to obtain tube bottom precipitate which is Scolopendra mutilans venom gland total RNA.

II, purifying the centipede mutilans venom gland mRNA:

the mRNA of the poisonous gland of the scolopendra mutilans is separated and purified by PROMEGA company of the United states

mRNAamong Systems kit.

Dissolving 500 μ g of total RNA of scolopendra mutilans venom gland in 500 μ l of DEPC water, placing in 65 ℃ water bath for 10 minutes, adding 3 μ l of oligo (dT) probe and 13 μ l of 20 XSSC solution, mixing well, standing at room temperature and cooling to obtain solution A.

Washing of magnetic beads (SA-PMP): and (3) flicking and mixing the magnetic beads evenly until the mixture is adsorbed by a magnetic frame for 30 seconds, removing the supernatant, adding 0.5 XSSC 0.3mL until the mixture is adsorbed by the magnetic frame for 30 seconds, and finally adding 0.1ml of 0.5 XSSC for suspension, wherein the suspension is called liquid B.

Adding the solution A into the solution B, standing at room temperature for 10 minutes until the solution A is adsorbed by a magnetic rack for 30 seconds, discarding the supernatant, washing with 0.1 XSSC for 4 times, finally discarding the supernatant, adding 0.1mL of DEPC water for suspension, adsorbing on the magnetic rack for 30 seconds, transferring the supernatant to a new test tube, adding 0.15mL of DEPC water for resuspension, adsorbing on the magnetic rack for 30 seconds, transferring the supernatant to the test tube, and obtaining purified venomous scolopendra mRNA in the supernatant.

1/10 volumes of 3M sodium acetate, pH 5.2, equal volume of isopropanol were added, left at-70 ℃ for 30 minutes, centrifuged at 12000rpm for 10 minutes at 4 ℃, the supernatant discarded and the pellet dissolved in 10. mu.l of DEPC water.

III, constructing a centipede mutila venom gland cDNA library: a Kit was constructed using the plasmid cDNA library of the CreatorTM SMART TM cDNAlibrary Construction Kit of CLONTECH.

First strand cDNA Synthesis (reverse transcription of mRNA):

mu.l of Scolopendra mutilans venom gland mRNA, 1. mu.l of SMART IV oligonucleotide, 1. mu.l of CDS III/3' PCR primer, and 2. mu.l of deionized water were added to a 0.5ml sterile centrifuge tube to make the total volume 5. mu.l.

Mix the reagents in the centrifuge tube and centrifuge, and keep the temperature at 72 ℃ for 2 minutes. The centrifuge tubes were incubated on ice for 2 minutes. The following reagents 2.0. mu.l of 5 Xprimary strand buffer, 1.0. mu.l of 20mM Dithiothreitol (DTT), 1.0. mu.l of 10mM dNTP mix, and 1.0. mu.l of PowerScript reverse transcriptase were added to the centrifuge tube.

Mix the reagents in the centrifuge tubes and centrifuge and incubate at 42 ℃ for 1 hour.

The first strand synthesis was stopped by placing the centrifuge tube on ice.

Mu.l of the first strand of the synthesized cDNA was taken from the centrifuge tube and used.

B. Amplification of the second Strand Using Long-terminal polymerase chain reaction (LD-PCR)

The PCR instrument was preheated to 95 ℃.

Mu.l of first strand cDNA (reverse transcription of mRNA), 80. mu.l of deionized water, 10. mu.l of 10 × Advantage 2 PCR buffer, 2. mu.l of 50 × dNTP mix, 2. mu.l of 5 'PCR primer, 2. mu.l of CDS III/3' PCR primer, and 2. mu.l of E.coli polymerase centrifuge tube were reacted.

Amplification was performed in a PCR instrument according to the following procedure:

① 95 deg.C for 20 seconds

② 22 cycles:

95 ℃ for 5 seconds

68 ℃ for 6 minutes

After the circulation was completed, the double strand cDNA synthesized in the centrifuge tube was extracted.

PCR products from PROMEGA

The SV Gel and PCR Clean-Up System kit is extracted and recovered, and the steps are as follows:

1. adding cDNA double chains obtained by PCR into equal-volume membrane binding buffer, reversing and uniformly mixing, transferring the mixed solution into a centrifugal purification column, and standing at room temperature for 5 minutes to ensure that the DNA is fully bound with a silica gel membrane. Centrifuge at 16,000g for 1 minute and discard the tube.

2. Add 700. mu.l of eluent (containing ethanol) to the centrifugation and purification column, centrifuge for 1 minute at 16,000g, and discard the waste liquid from the collection tube.

3. And (5) repeating the step (2).

4.16,000 g were centrifuged for 5 minutes.

5. The centrifugal purification column was placed in a new centrifuge tube.

6. 30. mu.l of ultrapure water was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes.

7.16,000 g, and centrifuging for 1 minute to obtain the bottom solution of the tube which is the purified cDNA double strand.

D. Preparation of E.coli DH5 α competent cells:

1. a single DH5 α colony was picked and inoculated into 3mL LB medium without ampicillin and cultured overnight at 37 ℃, the bacterial suspension was re-inoculated into 50mL LB medium the next day at a ratio of 1: 100 and shaken at 37 ℃ for 2 hours, and when the OD600 reached 0.35, the bacterial culture was harvested.

2. The bacteria were transferred to a sterile, single-use, ice-pre-chilled 50mL polypropylene tube and placed on ice for 10min and the culture cooled to 0 ℃.

3. The cells were recovered by centrifugation at 4100r/min for 10min at 4 ℃.

4. The broth was decanted and the tube inverted for 1min to allow the last traces of broth to drain.

5. Each cell pellet was resuspended in 50ml of initial broth and 30ml of a pre-cooled 0.1mol/L CaCl2-MgCl2 solution (80mmol/LMgCl2, 20mmol/LCaCl 2).

6. The cells were recovered by centrifugation at 4100r/min for 10min at 4 ℃.

7. The broth was decanted and the tube inverted for 1min to allow the last traces of broth to drain.

8. Each 50mL of the initial culture was resuspended in 2mL of ice-chilled 0.1mol/L CaCl2 and the cell pellet was aliquoted.

E. Enzyme cutting, connection and transformation of connection products:

1. mu.l of TakarapMD18-T vector and 4. mu.l of scolopendra mutilans cDNA double-stranded solution are added into a microfuge tube, and the total amount is 5. mu.l.

2. Mu.l (equal amount) of ligase buffer mixture was added.

The reaction was carried out at 3.16 ℃ for 2 hours.

4. The total amount (10. mu.l) was added to 100. mu.l of DH5 α competent cells and placed on ice for 30 min.

After heating at 5.42 ℃ for 90 seconds, it was left on ice for 1 minute.

6. Then, 890. mu.l of LB medium incubated at 37 ℃ was added thereto, and the mixture was incubated at 37 ℃ for 60 minutes with slow shaking.

7. 200. mu.l of the suspension was spread on LB medium containing X-Gal, IPTG and Amp and cultured at 37 ℃ for 16 hours to form a single colony.

8. Colonies were washed with 5mL LB liquid medium per LB plate and frozen with 30% glycerol. The constructed cDNA contained approximately 1X 10⁶Individual clones.

Centipede polypeptide SLP _ SsTx gene cloning:

the first degenerate primer (5 'GARGTNATHAARAARGAYACN 3', 5 'AAGCAGTGGTATCAACGCAGAGTACT 3') was used to amplify the mature peptide portion of the centipede polypeptide SLP _ SstX, and the second degenerate primer (5 'CGTTTTTGAAAAGTTGTAGTA 3', 5 'TGTTATTTTACCACTGGTTAA 3') was used to amplify the signal peptide portion of SLP _ SstX.

The complete sequence of the cDNA encoding the centipede polypeptide SLP _ SstX was thus obtained:

the PCR reaction was performed under the following conditions: 30 seconds at 94 ℃, 30 seconds at 60 ℃ and 45 seconds at 72 ℃ for 35 cycles.

Example 2

Separation and purification of centipede polypeptide SLP _ SstX:

sephadex G-50 gel filtration chromatography:

dissolving 200mg of lyophilized Scolopendra venom in 20ml of 0.1M phosphate buffer (pH6.0), centrifuging at 12000rpm for 10min, collecting supernatant, purifying with Sephadex G-50 gel filtration column (26 × 100cm), eluting with the same buffer, collecting with automatic fraction collector at flow rate of 3 ml/tube/10 min, detecting protein or polypeptide concentration in the collected solution with 280nm ultraviolet, mixing fractions with analgesic activity, and lyophilizing at-20 deg.C.

Reverse phase high pressure liquid chromatography (RP-HPLC):

the active peak obtained by Sephadex G-50 gel filtration chromatography was redissolved in 2ml of 0.1M phosphate buffer (pH6.0), centrifuged at 12000rpm for 15 minutes at 4 ℃ to obtain a supernatant, which was then filtered through a 0.45-. mu.m filter, and the filtrate was collected and applied to a reversed-phase high-pressure liquid phase C18 column, and gradient eluted at 0.7ml/min using an elution system composed of water (containing 0.1% trifluoroacetic acid) and acetonitrile (containing 0.1% trifluoroacetic acid). Collecting Scolopendra polypeptide SLP _ SstX, lyophilizing, and storing at-20 deg.C. The purified centipede polypeptide SLP _ SstX was subjected to N-terminal sequencing by Edman degradation (model 491, ABI). The polypeptide SLP _ SstX molecular weight was determined by flight mass spectrometry (MOLIDI-TOF).

Example 3

Chemical synthesis and tertiary structure determination of centipede polypeptide SLP _ SstX:

I. chemical synthesis method of centipede polypeptide SLP _ SstX: the complete sequence was synthesized by an automated polypeptide synthesizer based on amino acid sequencing of the encoded centipede polypeptide SLP _ SstX. Desalting by HPLC reverse phase C18 column chromatography, and purifying.

Molecular weight and purity identification of purified centipede polypeptide SLP _ SstX, analysis was performed using flight mass spectrometry (MOLDI-TOF) and reverse phase high-phase liquid chromatography (RP-HPLC).

III.600MHz high field nuclear magnetic resonance (600-MHz Bruker AV600) was used to study the tertiary structure of the centipede polypeptide SLP _ SstX. Sample preparation conditions were 500. mu.l 4mM SLP _ SstX, solution conditions 90% PBS/10% D2O, pH 6.5. All nuclear magnetic data obtained were analyzed with NMRPipe/NMRDraw software and spark program. PyMol was used to show the final three-dimensional structure.

The centipede polypeptide SLP _ SstX is a single-chain polypeptide encoded by centipede oligochaeta gene and has the molecular weight of 6017.6 daltons. The full length of the protein sequence is as follows: EVIKKDTPYKKRKFPYKSECLKACATSFTGGDDSRIQEGKPGFFKCTCYFTTG (a in fig. 1). The tertiary structure shows that SLP _ SstX is a polypeptide toxin (B in figure 1) with compact structure formed by two pairs of intramolecular disulfide bonds, the N-terminal presents flexible structure, and the C-terminal structure is conserved (C in figure 1).

Example 4

Pharmacological function of the centipede polypeptide SLP _ SsTx:

KCNQ channel inhibitor activity:

HEK293 cells transiently overexpressing mouse KCNQ channels were used to study the interaction between SLP _ SsTx and KCNQ family channels.

Cells with smooth cell membranes and uniform cytoplasm are selected under an inverted microscope, and the patch clamp experiment is carried out at the room temperature of 20-25 ℃. The method comprises the steps of selecting a WPI 0.86mm thin-wall borosilicate glass capillary tube as a glass electrode material, drawing the glass electrode on a drawing instrument (P-97, Shutter) for 5 steps, wherein the caliber of the tip of the electrode is 1.5-3.0 mu m after the glass electrode is subjected to thermal polishing, and filling intracellular fluid into the glass electrode after drawing is finished. The initial resistance of the glass electrode is preferably 1.5 to 2.5 M.OMEGA.. And after the electrode and the cell membrane form high-impedance Jingou (G omega) sealing, the electrode fast capacitor is supplemented. The cells were then clamped at-60 mV and a short, powerful negative pressure was applied to rapidly break the clamped cell membrane in the electrode, again compensating for the cell's slow capacitance. After the whole cell recording mode was established, the cells were clamped to 0mV and the cells stabilized for 10 minutes and the recording of current was started using the appropriate pulse voltage (80/-80 mV). The drugs were perfused using Biolab RS200 with a switching speed between drugs of 50 ms. The series resistance (Rs) is kept constant in the range of 5-8M omega all the time during the experiment, and the system series resistance compensation is generally between 30-60%. The experimental data were analyzed using Patch Master software and further analysis of the data was performed using Igor software. All results are expressed as mean (Average) ± Standard Error (SEM), and n represents the number of data tested.

SLP _ SsTx inhibited KCNQ4 channel in concentration dependence (a in fig. 2), with an IC50 of 2.52 ± 0.17 μ M (n ═ 10) (B in fig. 2).

SLP _ SsTx affects thoracic aortic vasoconstriction by inhibiting KCNQ channels.

In vivo effects of Sstx and centipedes on the cardiovascular system: under the effect of 5mg/ml and 10mg/ml toxins, respectively, the systolic force of coronary artery increased by about 1.6 (80%) and about 2.1g (105%), respectively, while the positive control group saturated phenylephrine increased by about 2.2g (110%) (a in fig. 3). To determine whether SstX is a key component of the venom that causes vascular disorders, we compared the vascular activity of crude toxicity of SstX presence and absence (cv-SstX free, cv-SF). As shown by B in fig. 3, the crude venom significantly reduced systolic arterial activity in the absence of SsTx. Compared with the 10mg/ml crude toxin-induced vasoconstriction response, 10mg/ml cv-SF increased only about 7.5% of vasoconstriction, indicating that SstX is the major active ingredient affecting vasoactivity. As expected, 1. mu.M and 5. mu.M SstX increased the contractile force of the coronary arteries by about 50.3 and about 105%, respectively (C in FIG. 3). Based on the biological mechanisms of SstX, retigabine significantly reduced centipede neurotoxicity and SstX-induced vascular toxicity (D-F in FIG. 3).

Inhibition of KCNQ family channels by SLP _ SsTx leads to cardiovascular diseases:

as expected, Intravenous (IV) injection of crude centipede venom resulted in acute hypertension in mice (a in fig. 4). In addition, 1.0mg/kg of Sstx further caused a significant increase in blood pressure in mice [ systolic blood pressure increased by 30.4%, diastolic blood pressure increased by 40 ± 6.3% ] (B in FIG. 4). Like mice, SsTx caused vasospasm and acute hypertension in rhesus macaques, while atropine and retigabine inhibited SsTx activity (C & D in figure 4). Intravenous retigabine (1mg/kg) reversed SstX induced hypertension (0.1mg/kg) with a drop in blood pressure from-196 mmhg to the normal range (D in FIG. 4). After SstX injection, mice continued to twitch and shudder before death, with LD50 of-0.85 mg/kg, and an elimination half-life of 4.5 h. This lethal activity was probably due to myocardial ischemia caused by coronary arteries, since we observed inverted T-waves (E in fig. 4) of monkeys 5 minutes after intravenous injection of SsTx. T-wave inversion is a typical representation of myocardial ischemia syndrome, whereas retigabine reverses T-wave inversion (E in fig. 4). Thus, Sstx-induced coronary spasm plays a key role in myocardial ischemia, and if ischemic symptoms are prolonged, heart failure often progresses.

SLP _ SsTx inhibition of KCNQ family channels affects the nervous system and respiratory system:

the neural KCNQ channels (including KCNQ2, 3 and 5) are the molecular basis for M currents. A 25% reduction in M current can lead to neonatal epilepsy in humans, while KCNQ2 knockout in mice is fatal. Our studies showed that SsTx is a potent inhibitor of KCNQ2, KCNQ3, and KCNQ5 (B in fig. 2). Therefore, we concluded that injection of SsTx in the hippocampus of animals may cause severe neurological disorders. Mice developed cramps immediately after injection of 1 μ l of 20 μ l of SstX into the hippocampus, and died after 10-20 minutes. We further recorded mouse brain slices. At a concentration of 10 μ M Sstx, the Sstx-induced pulse frequency was-20 ("-represents approximately"), significantly higher than that of untreated neurons (-8) (A & B in FIG. 5). After treatment with 1 μ l of 10 μ M SstX and 100 μ M linopridine, a known KCNQ channel inhibitor, mouse hippocampal acetylcholine concentrations increased by-63.6% and 81.8%, respectively (C in FIG. 5).

In view of the important role of KCNQ in the respiratory system, the effects of rough toxicity and SsTx on rat respiratory function were subsequently investigated (D in fig. 5). After intravenous injection of Sstx, the respiratory frequency is significantly reduced and the respiratory amplitude is increased. 1.5 hours after administration of 2mg/kg Sstx, respiration rate decreased 75% and respiration amplitude increased 150% (D-F in FIG. 5). The bronchoconstriction (G & H in fig. 5) by rough toxin and SsTx is again blocked by retigabine. Our results indicate that crude toxicity of SsTx and centipedes causes disorders not only to the cardiovascular system, but also to the nerves and respiratory system.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Kunming animal research institute of Chinese academy of sciences

<120> centipede polypeptide SLP _ SstX and coding gene and application thereof

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Glu Val Ile Lys Lys Asp Thr Pro Tyr Lys Lys Arg Lys Phe Pro Tyr

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Lys Ser Glu Cys Leu Lys Ala Cys Ala Thr Ser Phe Thr Gly Gly Asp

20 25 30

Asp Ser Arg Ile Gln Glu Gly Lys Pro Gly Phe Phe Lys Cys Thr Cys

35 40 45

Tyr Phe Thr Thr Gly

50

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atttcaactg aagttattaa aaaagatact ccatataaaa aaagaaaatt tccttataaa 120

agtgagtgtt tgaaggcctg cgcaacttct tttacaggag gagatgaaag tagaatacaa 180

gaaggaaaac ctgggttctt caaatgtacc tgttatttta ccactggt 228

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gaagttatta aaaaagatac tccatataaa aaaagaaaat ttccttataa aagtgagtgt 60

ttgaaggcct gcgcaacttc ttttacagga ggagatgaaa gtagaataca agaaggaaaa 120

cctgggttct tcaaatgtac ctgttatttt accactggt 159

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gargtnatha araargayac n 21

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<213> Artificial Sequence (Artificial Sequence)

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aagcagtggt atcaacgcag agtact 26

<210>6

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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cgtttttgaa aagttgtagt a 21

<210>7

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<213> Artificial Sequence (Artificial Sequence)

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tgttatttta ccactggtta a 21

Claims

1. A centipede polypeptide SLP _ SstX for inhibiting KCNQ family channels is characterized in that the whole amino acid sequence of the centipede polypeptide SLP _ SstX is shown as SEQ ID NO. 1.

2. The gene for coding the centipede polypeptide SLP _ SstX as claimed in claim 1, which comprises a mature peptide part corresponding gene and a signal peptide part corresponding gene, and the nucleotide sequence of the centipede polypeptide SLP _ SstX gene is shown in SEQ ID NO. 2.

3. The gene as claimed in claim 2, wherein the nucleotide sequence of the corresponding gene of the centipede polypeptide SLP _ SstX mature peptide part is shown in SEQ ID No. 3.

4. The method for amplifying the gene of the centipede polypeptide SLP _ SstX as claimed in claim 2, which comprises the following steps:

2) purifying mRNA from the total RNA obtained in the step 1) to obtain mRNA;

4) amplifying a corresponding gene of a centipede polypeptide SLP _ SstX mature peptide part by using a first degenerate primer pair by taking a cDNA library as a template; amplifying a corresponding gene of the centipede polypeptide SLP _ SstX signal peptide part by using a second degenerate primer pair by taking the cDNA library as a template; obtaining the complete sequence of the gene of claim 2; the nucleotide sequence of the first degenerate primer pair is shown as SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the second degenerate primer pair is shown as SEQ ID NO.6 and SEQ ID NO. 7; wherein R ═ a or G, Y ═ C or T, N ═ A, C, G or T in the sequences of the first degenerate primer pair.

5. An inhibitor of KCNQ family channels comprising the centipede polypeptide SLP _ SsTx of claim 1 and an excipient.

6. The inhibitor of claim 5, wherein the excipient comprises a sterile aqueous solution of 0.9% sodium chloride.

7. The use of the centipede polypeptide SLP _ SstX of claim 1 in the preparation of a medicament for alleviating centipede bites.